Within the past decade, several technologies have made it possible to monitor the expression level of a large number of genetic transcripts at any one time (see, e.g., Schena et al., 1995, Science 270:467–470; Lockhart et al., 1996, Nature Biotechnology 14:1675–1680; Blanchard et al., 1996, Nature Biotechnology 14:1649; Ashby et al., U.S. Pat. No. 5,569,588, issued Oct. 29, 1996). For example, techniques are known for preparing microarrys of cDNA transcripts (see, e.g., DeRisi et al., 1996, Nature Genetics 14:457–460; Shalon et al., 1996, Genome Res. 6:689–645; and Schena et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 93:10539–11286). Alternatively, high-density arrays containing thousand of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ are described, e.g., Fodor et al., 1991, Science 251:767–773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022–5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270). Methods for generating arrays using inkjet technology for oligonucleotide synthesis are also known in the art (see, e.g., Blanchard, International Patent Publication WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996, Biosensors and Bioelectronics 11:687–690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages 111–123).
Applications of this technology include, for example, identification of genes which are up regulated or down regulated in various physiological states, particularly diseased states. Additional exemplary uses for transcript arrays include the analyses of members of signaling pathways, and the identification of targets for various drugs. See, e.g. Friend and Hartwell, International Publication No. WO 98/38329 (published Sep. 3, 1998); Stoughton, U.S. patent application Ser. No. 09/099,722 (filed Jun. 19, 1998); Stoughton and Friend, U.S. patent application Ser. No. 09/074,983 (filed May 8, 1998); Friend and Stoughton, U.S. Provisional Application Ser. Nos. 60/084,742 (filed May 8, 1998), 60/090,004 (filed Jun. 19, 1998), and 60/090,046 (filed Jun. 19, 1998).
However, several factors limit the number of genetic transcripts that can be detected on a single microarray “chip.” In particular, the “reporting density” (i.e., the number of genes detected per unit of surface area) for a microarray is limited, e.g., by the density with which polynucleotide probes may be laid down as well as by the number of polynucleotide probes required per gene. A plurality of probe pairs, which are both matched to and intentionally mismatched to a target sequence, are required in order to empirically distinguish signal arising from a target polynucleotide sequence of interest (e.g., a particular mRNA sequence of interest) from signal arising from cross-hybridization with other polynucleotide sequences. Currently, in situ synthesized microarray chips require more than 20 oligonucleotide probe pairs per gene or gene region reported (Lockhart et al., supra). On the other hand, the number of polynucleotide probes that may be laid down on a microarray chip is limited by the technology used to produce the microarray. Photolithographic techniques discussed above for producing oligonucleotide microarrays having a high spatial density of probes are expensive to synthesize and therefore require a large capital investment. Oligonucleotide microarrays produced using the above discussed inkjet technology methods are, by contrast, much cheaper and faster to produce both per chip design and per chip. Thus, such microarrays are generally preferred for detecting genetic transcripts in cells. However, microarray chips produced by such inkjet technology have a limited probe density that is only a fraction of the probe density of chips produced by photolithography methods. Thus, at present the number of genetic transcripts that may be detected on a single microarray chip is limited to about 10,000 gene transcripts using expensive, photolithographic arrays, and only about 750 to 2,500 gene transcripts using less expensive, inkjet arrays.
There exists therefore a need for materials and methods which may be used to efficiently detect large numbers of different genetic transcripts and thereby detect changes in a large number of genetic transcripts in a cell or cells. In particular, there is a need for materials and methods which may be used to detect changes in genetic transcription across the entire genome of a cell, including cells of complex organisms such as mammalian cells and, in particular, human cells.
There also exists, however, a need for materials and methods which may be used to accurately detect charges in genetic transcripts in cells, e.g., in response to some environmental change or perturbation. In particular, there is a need to accurately detect changes in the expression levels of those particular genetic transcripts that exhibit the largest changes, e.g., in response to an environmental change or perturbation, and which are therefore most relevant in understanding the effect of the environmental change or perturbation on the cell or cells.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.